Difference between revisions of "Team:JMU Wuerzburg/Description"

 
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            <h3>Test Tonic – a rapid test system for the malaria-causing parasite <span style="font-style: italic;">Plasmodium</span></h3>
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                Malaria is a widely spread infectious disease that causes
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                400.000 deaths every year and affects more than 200 million
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                people in total according to the WHO's Worlds Malaria Report 2017.
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                The disease is caused by different species of the protozoan parasite <i>Plasmodium</i>.
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                For a successful therapy of Malaria, a fast and sensitive
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                detection of the species affecting the patient is crucial.
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                <sup><a href="#desc_refs">1</a></sup>
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                <br><br>
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                Our aim is to construct a test system that is capable of
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                detecting the DNA of <i>Plasmodium</i>.
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                We venture to create a rapid, easily usable and cheap diagnostic
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                device for large area application. To reach this goal,
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                we design primers and probes by analysing published sequences
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                of the human pathogenic <i>Plasmodium</i> species with bioinformatic tools.
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                We reach out to create one primer-probe pair that can detect <i>Plasmodium</i>
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                in general and a specific primer/probe pair for <i>P. falciparum</i>.
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                <br><br>
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                For the amplification of characteristic parts of the <i>Plasmodium</i>
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                genome we developed two pairs of primers for each species.
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                One of these indicates if a patient is affected of <i>Plasmodium</i> in general.
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                The other one specifically detects the species <i>Plasmodium falciparum</i>.
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                <br><br>
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                After identification of fitting sequences,
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                we evaluate and optimize our primers by running qPCR assays with
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                E. coli containing our BioBrick: a plasmid with a short,
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                synthetic, non-pathogenic sequence of the <i>Plasmodium</i> genome
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                as a positive control. Additionally we conduct qPCR assays
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                with genomic DNA of cultured <i>Plasmodium</i> parasites.
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                <br><br>
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                To perform the amplification directions in a user-friendly
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                way we designed a hardware model. The reactions steps are conducted
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                in a simple test tube with separated chambers to isolate different reactions.
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                A step motor and an Arduino manage the movement and mixing of the reaction fluids.
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                <br><br>
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                After creating a fundamental model with a multiplex qPCR we elaborate
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                a way to apply our detection system to Recombinase Polymerase Amplification
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                (RPA)<sup><a href="#desc_refs">2</a></sup>,
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                <sup><a href="#desc_refs">3</a></sup>. RPA is a promising alternative for qPCR
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                to isothermally amplify our target sequences in a short period of time.
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                It has been shown to be a sufficient method to detect
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                Plasmodium DNA by Kersting et al. in 2014<sup><a href="#desc_refs">4</a></sup>.
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                RPA makes such a test system cheap and avoids the
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                need of an expensive thermocycler. This results in many advantages
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                for the application in travelling situations and in areas without
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                proper infrastructure and energy supply.
  
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        <title>iGEM Würzburg - Project Description</title>
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                <h5 id="collabs_refs">List of References</h5>
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                <sup>1</sup> <a href="http://www.who.int/malaria/areas/treatment/overview/en/">http://www.who.int/malaria/areas/treatment/overview/en/</a><br>
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                <sup>2</sup> <a href="https://www.ncbi.nlm.nih.gov/pubmed/27160000">https://www.ncbi.nlm.nih.gov/pubmed/27160000</a><br>
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        <h2>Development of a qPCR-based rapid testen system</h2>
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                <sup>3</sup> <a href="https://reader.elsevier.com/reader/sd/pii/S0165993617302583?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F">https://reader.elsevier.com/reader/sd/pii/S0165993617302583</a><br>
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        <p>Recently, fast qPCR and RTqPCR diagnostical tests detecting pathogens in patient-derived samples have been developed. These tests comprise all required steps from genome purification to the result analyses in a "single tube" reaction. Our aim is to construct a test system that is adaptable on various pathogens. At first we design primers and probes by analysing all published sequences of the respective pathogens with bioinformatic tools. After that we develop our rapid tests detecting Noroviruses and the malaria-causing pathogens Plasmodium. Our test system is evaluated and optimised using synthesised templates. In a second step, we use inactivated patient-derived samples to confirm selectivity and specificity of our tests.
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                <sup>4</sup> <a href="https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99">https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99</a>
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Latest revision as of 15:31, 17 October 2018

Test Tonic – a rapid test system for the malaria-causing parasite Plasmodium

Malaria is a widely spread infectious disease that causes 400.000 deaths every year and affects more than 200 million people in total according to the WHO's Worlds Malaria Report 2017. The disease is caused by different species of the protozoan parasite Plasmodium. For a successful therapy of Malaria, a fast and sensitive detection of the species affecting the patient is crucial. 1

Our aim is to construct a test system that is capable of detecting the DNA of Plasmodium. We venture to create a rapid, easily usable and cheap diagnostic device for large area application. To reach this goal, we design primers and probes by analysing published sequences of the human pathogenic Plasmodium species with bioinformatic tools. We reach out to create one primer-probe pair that can detect Plasmodium in general and a specific primer/probe pair for P. falciparum.

For the amplification of characteristic parts of the Plasmodium genome we developed two pairs of primers for each species. One of these indicates if a patient is affected of Plasmodium in general. The other one specifically detects the species Plasmodium falciparum.

After identification of fitting sequences, we evaluate and optimize our primers by running qPCR assays with E. coli containing our BioBrick: a plasmid with a short, synthetic, non-pathogenic sequence of the Plasmodium genome as a positive control. Additionally we conduct qPCR assays with genomic DNA of cultured Plasmodium parasites.

To perform the amplification directions in a user-friendly way we designed a hardware model. The reactions steps are conducted in a simple test tube with separated chambers to isolate different reactions. A step motor and an Arduino manage the movement and mixing of the reaction fluids.

After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA)2, 3. RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. It has been shown to be a sufficient method to detect Plasmodium DNA by Kersting et al. in 20144. RPA makes such a test system cheap and avoids the need of an expensive thermocycler. This results in many advantages for the application in travelling situations and in areas without proper infrastructure and energy supply.
List of References
1 http://www.who.int/malaria/areas/treatment/overview/en/
2 https://www.ncbi.nlm.nih.gov/pubmed/27160000
3 https://reader.elsevier.com/reader/sd/pii/S0165993617302583
4 https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99